Studies on Acaricidal Bioactivities of Artemisia annua L.Extracts Against Tetranychus cinnabarinus Bois.(Acari:Tetranychidae)

The aim of this study was to determine the best extraction technique, the most suitable solvent, the optimal plant parts, and the acaricidal activities of Artemisia annua L. The petroleum ether （30-60℃）, petroleum ether （60-90℃）, ethanol, acetone, and water parallel and sequenced extracts were obtained from the leaves, stems and roots of different period of A. annua L. in April, May, June, July and September respectively. And then the acaricidal bioactivities against Tetranychus cinnabarinus of all extracts were determined by the slide-capillary method in the laboratory. The results indicated that the acaricidal bioactivities elevated as the development of A. annua plant at the concentration of 5 mg mL-L The general tendency exhibited the sequence of July 〉 June 〉 May 〉 April, but September decreased comparing to July. However, the most effective extracts in five months were all acetone parallel extract of A. annua leaf, and the corrected mortalities treated after 48 h ranged from 74 to 100%. The median lethal concentrations （LC50） against T. cinnabarinus of acetone parallel extracts ofA. annua leaves in September, July, June, May and April were 0.5986, 0.4341, 0.8376, 0.9443 and 1.3817 mg mL^-1, respectively, treated after 48 h. The 13 groups were isolated from acetone extracts ofA. annua leaves in July by column chromatography, both the 1 lth and 12th groups exhibited strong bioactivities. The median lethal concentrations of the 1 lth and 12th groups against T. cinnabarinus were 0.3683 and 0.1586 mg mL^-1, respectively. The acetone parallel extract ofA. annua leaf in July was the most toxic to T. cinnabarinus and the corrected mortality was 100% after 48 h. The acetone parallel extract of the 1 lth and 12th groupswere the most active components, acted as the emphases in further study.

Resistance Selection of Tetranychus cinnabarinus to Three Acaricides and Its Management Strategy

The effects of retarding resistance evolution of mixing and sequencing acaricides were studied based on the resistance selection of three acaricides to Tetranychus cinnabarinus in the laboratory. Tetrany-chus cinnabarinus was continuously selected with fenpropathrin, abamectin and pyridaban for 28, 30 and 31 generations, respectively, and resistance factor increased to 65.6, 7.2 and 1.2 time, accordingly. Results of cross-resistance determination suggested that there were no cross-resistance existed among the three acaricides. Analysis of resistance selection and estimation of realized heritability (h2) of resistance to three single and two mixed acaricides showed that a mixture of fenpropathrin+abamectin would slow resistance development compared with a sequence of fenpropathrin followed by abamectin while the mixture of pyridaben+abamectin and the sequence of pyridaben followed by abamectin would effectively retard resistance evolution.

Resistance Selection of Tetranychus cinnabarinus to Fenpropathrin and Genetic Analysis

The resistant inheritance mode of Tetranychus cinnabarinus to fenpropathrin were studied based on the resistance selection in laboratory. After 40 generations selection, T. cinnabarinus developed 68.5-fold resistance to fenpropathrin. The methods of cross and back-cross between resistant （R） and sensitive （S） strains were used for exploring the inheritance mode of the resistance of this mite to fenpropathrin. The log （Lc）-p equation of F1 got from obverse （SR） and reverse （RS） crosses was intermediate between S and R and inclined to S, the dominant indices DSR and DRS were -0.83 and -0.29, respectively, which indicated that the resistance is controlled by the incompletely recessive gene; the 95% confidence limit of this two dominant indices do not superpose showing DSR and DRS have significant difference and the maternal or external karyon effect may be existed in resistance inheritance; the back-cross results of heterozygote F1 with its parents suggested that the separation of progenies F2 （SR♀× S ♂ and RS ♀ × R ♂） was consistent with Mendel＇s single gene inheritance model.

The influence of Tetranychus cinnabarinus-induced plant defense responses on Aphis gossypii development

Carmine spider mites （Tetranychus cinnabarinus） and cotton aphids （Aphis gossypii） are both serious pests of cotton, and cause reductions in yields of this key agricultural crop. In order to gain insights into how plant defense responses induced by one herbivore species affect the behavior and performance of another, we examined how infestation with T. cinnabarinus influences the development of A. gossypfi using cotton as a model. In this study, we measured the activities of several important biochemical markers and secondary metabolites in the leaves of cotton seedlings responding to infestation by T. cinnabarinus. Furthermore, the influences of T. cinnabarinus infestation on the development ofA. gossypfi in cotton were also examined. Our data showed that the activities of several key defense enzymes, including phenylalanine ammonia-lyase （PAL）, peroxidase （POD）, lipoxygenase （LOX）, and polyphenol oxidase （PPO）, were substantially increased in cotton seedlings responding to spider mite infestation. Further, the contents of gossypol and condensed tannins, key defensive compounds, were significantly enhanced in leaves of cotton seedlings following T. cinnabarinus infestation. Moreover, the T. cinnabarinus-induced production of defense enzymes and secondary metabolites was correlated with infestation density. The developmental periods of A. gossypii on cotton seedling leaves infested with T. cinnabarinus at densities of 10 and 15 individuals cm-2 were 1.16 and 1.18 times that of control, respectively. Meanwhile, the mean relative growth rates of A. gossypfi on cotton leaves infested with T. cinnabarinus at densities of 8, 10 and 15 individuals cm-2 were significantly reduced. Therefore, these data suggested that the developmental periods of A. gossypfi were significantly lengthened and the mean relative growth rates were markedly reduced when cotton aphids were reared on plants infested with high densities of spider mites. This research sheds light on the role that inducible defense responses played in plant-mediated interspecific interactions between T. cinnabarinus and A. gossypfi.

Identification of the key chitinase genes in Tetranychus cinnabarinus (Boisduval) based on the expression and sequence characteristic analysis

Chitin is an important content in the exoskeletons of arthropods, and its hydrolyzation is catalyzed by chitinases during the process of molting, thus, the chitinases are considered as ideal target to interfere the growth of arthropods. This study intends to clarify the characteristic of the chitinases during the development of Tetranychus cinnabarinus, and screen out important genes as potential control targets. The results showed that the total enzyme concentration of chitinases was significantly higher in larva, the first and second nymph than that in egg and adult. Base on the transcriptome data, six unigenes encoding chitinases were identified and their expression patterns in different developmental stages were detected. The expressions of TcCHIT1 and TcCHITIO showed high abundance during the molting process and their expression change during the developmental stages was consistent with the enzyme concentration. The full-length of these two genes were further cloned, and the structural characteristics of their proteins were analyzed by constructing the three-dimensional structure model. The results provide basic information to understand the characteristic of chitinases in T. cinnabarinus and might be considered as target for control.

Integrated Control of Tetranychus cinnabarinus by Five Kinds of Pesticides Combined with Neoseiulus cucumeris

To find the method for the integrated control of Tetranychus cinnabarinus （carmine spider mite） and delay its resistance to pesticides, the joint actions of each of five commonly pesticides （abamectin, azadirachtin, matrine, pyrethrins and imidacloprid）, and Neoseiulus cucumeris （Oudemans）, the natural enemy of cotton red spider mite, were studied. N. cucumeris was released after application of pesticides for seven days （for 0,3% azadirachtin EC） or six days （for other pesticides）. The results showed that the combined action of 1.8% abamectin EW （1：8 000） and N. cucumeris had the best control efficacy of 96.63% at 20 days after N. cucumeris releasing. The control efficacies of N. cucumeds and 0.3% azadirachtin EC （1：250） were 59.7% and 90.1% after one day and 20 days, respectively, after N. cucumeris releasing. The control efficacy of N. cucumeris and 0.5% matrine AS （1：2 000） was 82.65% at 20 days after N. cucumeris releasing. The results provide options for sustainable control of T. cinnabarinus and for the delay of pesticides resistance.

Joint Acaricidal Action of Diafenthiuron and Fenbutatin-oxide Against Tetranychus cinnabarinus(Boisduval)(Acari: Tetranychidae)

[Objective]This study was conducted to obtain a new high-efficiency acaricide mixture.[Method]The synergism acaricidal of diafenthiuron and fenbutatin-oxide against carmine spider mite was investigated,and the optimal ratio of both acaricides were determined by means of co-toxicity factors and co-toxicity coefficients(CTC).[Result]At 24 h post-treatment,the LC50values of diafenthiuron and fenbutatin-oxide against T.cinnabarinus female adults were 154.67 and 93.26 mg/L,respectively,and the synergistic mass ratios of diafenthiuron and fenbutatin-oxide ranged from 1:0.06 to 1:0.87,and the optimal diafenthiuron-to-fenbutatin-oxide ratio was 1:0.39 with the LC50and CTC of 67.87 mg/L and 188.93(CTC significantly above 100 strongly indicated synergism),respectively,and the toxicity relative to diafenthiuron and fenbutatin-oxide was 2.28 and 1.37 times,respectively.[Conclusion]The joint acaricidal action of diafenthiuron and fenbutatin-oxide strongly indicates synergism.These findings proved scientific theoretical basis for mixing diafenthiuron and fenbutatin-oxide to control spider mites and developing a new synergisic acaricide.

Biochemical Evidences for Scopoletin Inhibits Ca^(2+)-ATPase Activity in the Carmine Spider Mite,Tetranychus cinnabarinus(Boisduval)

[Objective] This study aimed to investigate the acaricidal lffect of scopo- letin, and provide the biochemical evidences of scopoletin influencus Ca2＋-ATPase activity and gene expressions in the Carmine Spider Mite, Tetranych＇us cinnabarinus. in increased protein contents; kinetic analysis showed that the catalytic capability of Ca2＋-ATPase was significantly reduced by scopoletin. [Conclusion] Scopoletin exhibits a significant inhibitory effect on Ca2＋-ATPase, and its acaricidal effect against T. cinnabarinus might be due to the direct inhibition of Ca2＋-ATPase.

The dynamic changes of genes revealed that persistently overexpressed genes drive the evolution of cyflumetofen resistance in Tetranychus cinnabarinus

Changes in gene expression are associated with the evolution of pesticide resis-tance in arthropods.In this study,transcriptome sequencing was performed in 3 different resistance levels(low,L;medium,M;and high,H)of cyflumetofen-resistant strain(YN-CyR).A total of 1685 genes,including 97 detoxification enzyme genes,were upregulated in all 3 stages,of which 192 genes,including 11 detoxification enzyme genes,showed a continuous increase in expression level with resistance development(L to H).RNA in-terference experiments showed that overexpression of 7 genes(CYP392A1,TcGSTd05,CCE06,CYP389A1,TcGSTz01,CCE59,and CYP389C2)is involved in the development of cyflumetofen resistance in Tetranychus cinnabarinus.The recombinant CYP392A1 can effectively metabolize cyflumetofen,while CCE06 can bind and sequester cyflumetofen in vitro.We compared 2 methods for rapid screening of resistance molecular markers,in-cluding short-term induction and 1-time high-dose selection.Two detoxification enzyme genes were upregulated in the field susceptible strain(YN-S)by induction with 20%lethal concentration(LC2o)of cyflumetofen.However,16 detoxification enzyme genes were up-regulated by 1-time selection with LCso of cyflumetofen.Interestingly,the 16 genes were overexpressed in all 3 resistance stages.These results indicated that 1685 genes that were upregulated at the L stage constituted the basis of cyflumetofen resistance,of which 192 genes in which upregulation continued to increase were the main driving force for the de-velopment of resistance.Moreover,the 1-time high-dose selection is an efficient way to rapidly obtain the resistance-related genes that can aid in the development of resistance markers and resistance management in mites.

ESTMATION OF REALIZED HERITABILITY OF RESISTANCE TO FENPROPATHRIN,ABAMECTIN,PYRIDABEN AND THEIR MIXTURES IN ACARICIDE-SELECTED STRAINS OF TETRANYCHUS CINNABARINUS (BOIDUVAL)

Threshold trait analysis was used to estimate realized heritability (h 2) of re sistance to five acaricides (three single acaricide and two mixtures) and resistance risk in Tetranychus cinnabarinus (Boiduval). Tetranychus cinnabarinus collected from the field of Beibei, Chongqing reared more than 60 generations under pesticide free conditions and considered s usceptible. Successively selected for about 30 generations, the strain had a 65.55-, 5.82 - , 1.23-, 5.20- and 1.42-fold increase in resistance to fenpropathrin, abam ectin, pyridaben, pyridab en-abamectin (pyridaben: abamectin=7.4:0.1, m/m) and fenpropathrin-abamectin (fenpropathri n: abamectin=8.9:0.1, m/m), respectively. The realized heritability of resista nce t o fenpropathrin, abamectin, pyridaben, pyridaben-abamectin (pyridaben: abamectin=7.4:0.1, m/m) and fenpropathrin-abamectin (fenpropathrin: abamectin=8.9:0.1, m/m) is 0.2167, 0 .0967, 0.0130, 0.0800 and 0.0172, respectively. Under the selected condition, a 10-fold incr ease in resistance would be expected 15 generations for fenpropathrin, 34 generations for abamectin , 333 generations for pyridaben, 42 generations for pyridaben-abamectin (pyridaben: a bamectin=7.4:0.1, m/m) and 200 generations for fenpropathrin-abametcin (fenpropathrin: abamectin= 8.9:0.1, m/m). The highest resistance risk of the five acaricides in Tetranychus cinnabarinus was fenpropathrin, then abamection, pyridaben-abamectin (pyridaben: abamectin=7.4:0.1, m/m), fen propathrin- abamectin (fenpropathrin: abamectin=8.9:0.1, m/m) and that to py ridaben was the lowest. The mixture of pyridaben and abamectin is not useful in delaying development of resistance in the pest to the two single acaricide while the mixture of fenpropathrin and abamectin could do it.

Molecular characterization of two novel esterase genes from carmine spider mite, Tetranychus cinnabarinus （Acarina： Tetranychidae）

Two novel esterase complementary DNAs were identified and cloned from the insecticide-susceptible strain of Tetranychus cinnabarinus （Boisduval） （Acarina： Tetrany- chidae）, which were designated as TCE1 and TCE2, respectively. The cDNA of TCE1 gene contained an open reading frame （ORF） of 1701 bp encoding 567 amino acids, and a predicted molecular weight of 62.75 kDa, the cDNA of TCE2 contained an ORF of 1680 bp encoding 560 amino acids, and a predicted molecular weight of 63.14 kDa. TCE1 and TCE2 were submitted to GenBank, accession number EU 130461 and EU 130462. The well-conserved sequence motif, GXSXG, used as a signature pattern in the esterase fam- ily are present in both TCE1 and TCE2 （GQSAG in TCE1, whereas GESAG in TCE2）, indicating that these two genes are predicted to be esterases. Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from Boophilus microplus showed that TCE1 shares 33.98% identity and TCE2 shares 33.46% identity. TCE1 and TCE2 share 46.4% identity. Quantitative real-time polymerase chain reaction revealed that expression level of the TCE2 gene was relatively higher than that of the TCE1 in all instars examined except the protonymph, and the expression level of these two esterase genes in adults of T. cinnabarinus was significantly higher than that in any other instars, respectively. T. cinnabarinus is an important agricultural mite pest and esterases are important in the metabolisms of insects and mites; the genomic information obtained in this study will contribute to esterase molecular biological study on mite pest species.

Transgenic cotton expressing C YP392A4 double-stranded RNA decreases the reproductive ability of Tetranychus cinnabarinus

As a polyphagous pest, Tetranychus cinnabarinus has the ability to overcome the defense of various hosts, and causes severe losses to various economically important crops. Since the interaction between pest and host plants is a valuable clue to investigate potential ways for pest management, we intend to identify the key genes of T. cinnabarinus for its adaption on cotton, then, with RNA interference （RNAi） and transgenic technology, construct a transgenic cotton strain to interfere with this process, and evaluate the effect of this method on the management of the mites. The difference of gene expression of T. cinnabarinus was analyzed when it was transferred to a new host （from cowpea to cotton） through high-throughput sequencing, and a number of differentially expressed genes involved in detoxification, digestion and specific processes during the development were classified. From them, a P450 gene CYP392A4 with high abundance and prominent over-expression on the cotton was selected as a candidate. With transgenic technology, cotton plants expressing double-stranded RNA of CYP392A4 were constructed. Feeding experiments showed that it can decrease the expression of the target gene, result in the reduction of reproductive ability of the mites, and the population of T. cinnabarinus showed an apparent fitness cost on the transgenic cotton. These results provide a new approach to restrict the development of mite population on the host. It is also a useful attempt to control piercing sucking pests through RNAi and transgenic technology.

Transcription factors CncC and Maf connect the molecular network between pesticide resistance and resurgence of pest mites

Pesticide resistance and resurgence are serious problems often occurring simultaneously in the field.In our long-term study of a fenpropathrin-resistant strain of Tetranychus cinnabaribus,enhancement of detoxification and modified fecundity mechanisms were both observed.Here we investigate the network across these two mechanisms and find a key node between resistance and resurgence.We show that the ecdysone pathway is involved in regulating the fecundity of T.cinnabaribus.The concentration change of ecdysone is consistent with the fecundity curve;the concentration of ecdysone is higher in the fenpropathrin-resistant strain which has stronger fecundity.The enhancement of ecdysone is due to overexpression of two P450 genes(CYP314A1 and CYP315A1)in the ecdysone synthesis pathway.Silencing expression of these CYP genes resulted in lower concentration of ecdysone,reduced expression of vitellogenin,and reduced fecundity of T.cinnabaribus.The expression of CYP315A1 is regulated by transcription factors Cap-n-collar isoform C(CncC)and Musculoaponeurotic fibrosarcoma protein(Maf),which are involved in regulating other P450 genes functioning in detoxification of fenpropathrin in T.cinnabaribus.A similar regulation is established in citrus pest mite Panonychus citri showing that the CncC pathway regulates expression of PcCYP315Al,which affects mite fecundity.Transcription factors are activated to upregulate detoxification genes facilitating pesticide resistance,while the“one to multiple”regulation mode of transcription factors simultaneously increases expression of metabolic enzyme genes in hormone pathways and alters the physiology of pests.This is an important response of arthropods to pesticides which leads to resistance and population resurgence.